Non-small cell lung cancer (NSCLC) remains a major challenge due to its resistance to apoptosis, limiting therapeutic success. Targeting survival pathways with Smac mimetics (e.g., Birinapant, Compound A) and BH3 mimetics (e.g., S63845, ABT-263) offers a promising approach. This study examines whether pre-treatment strategies enhance apoptosis compared to direct drug combination in NSCLC cells.

NSCLC cell lines were categorized into two groups based on apoptotic sensitivity. Group 1 (H727, LXF289, H358, CORL23, LCLC103H, SKMES1, H1299) responded to Smac mimetics alone, while Group 2 (A549, CALU1, COLO, H441, H2009, SKLU1) required additional BH3 mimetics. Group 1 cells were treated with Compound A, TNF-α, and caspase inhibitor QVD, and apoptosis was quantified via Annexin V/PI staining using flow cytometry. Necroptosis was assessed using RIPK1 and RIPK3 inhibitors (Necrostatin-1, GSK872) but did not significantly contribute to cell death.Group 2 cells underwent additional treatment with BH3 mimetics (S63845, ABT-199, WEHI-539, ABT-263). In one of sequential treatments, Birinapant was added first, followed by a 2-hour incubation before Smac mimetics, then TNF-α was introduced 30 minutes later. Group 1 cells were incubated for 24 hours, whereas Group 2 cells underwent 24-hour and 48-hour treatments to assess time-dependent effects.

Group 1 cells exhibited significant apoptosis with Smac mimetics alone, demonstrating reliance on IAP-mediated survival.Group 2 cells required BH3 mimetics for apoptosis, highlighting dependence on MCL-1/BCL-2 proteins.Sequential drug administration enhanced apoptosis, particularly in Group 2 cells. The sequential combination of Birinapant + S63845 + TNF-α was one of the most effective.Necroptosis inhibition did not alter cell death rates, confirming apoptosis as the dominant pathway. Extended 48-hour incubation further increased apoptosis in resistant cells.

Our findings highlight the importance of sequential drug administration in apoptosis induction. Smac mimetics effectively induced apoptosis in sensitive NSCLC cells, while resistant cells may require BH3 mimetics. The lack of necroptotic response confirms apoptosis as the primary mechanism. The common and possible genetic factors in the literature (e.g., mutant K-Ras, loss of p53, FHIT deletions) may contribute to therapy resistance. Optimizing pre-treatment regimens could enhance therapeutic efficacy in NSCLC patients.